Supra-physiological doses of anabolic androgenic steroids impact erythropoietin and blood parameters

Hematological parameters and erythropoiesis are known to be influenced by anabolic androgenic steroid (AAS) use. However, little is known in relation to supra-physiological doses of AAS. Therefore, the aim of this study was to evaluate the effect of supra-physiological doses of AAS on serum and urinary erythropoietin (EPO), and blood parameters, from self-reported AAS users. Serum EPO levels were higher in testosterone positive AAS users (11.83 mIU/mL ± 4.19) than nonpositive (6.60 mIU/mL ± 2.70, p = 0.03), while no differences in urinary EPO levels were noted. There were positive correlations between serum EPO and testosterone levels (rs = 0.46, p = 0.01) and reticulocyte percentage (rs = 0.43, p = 0.02). Individuals with AAS-induced hypogonadism (ASIH; luteinizing hormone levels <1.4 IU/L) had approximately 75% higher serum EPO (p < 0.05) and 140% higher high fluorescence reticulocyte fractions (p < 0.001), as well as other affected hematological parameters, compared with non-ASIH individuals. The results extend the knowledge of how endocrine and hematological biomarkers are affected by AAS doping.     

Detection of anabolic androgenic steroids in serum samples

When testing for anabolic androgenic steroids (AAS) outside sports communities, for example, in healthcare and forensic medicine, urine is the matrix of choice. However, there are drawbacks with urinary sampling, and serum might be useful as a complementary matrix. The aim was to develop an LC–MS/MS method for serum measuring AAS frequently used outside of sport, including testosterone (T), steroid esters, and eight other synthetic AAS. The sample pretreatment included sample precipitation and evaporation. Limit of quantification for the AAS was 0.05–0.5 ng/mL, and linearity was 0.05–20 ng/mL for most of the substances. Generally, the within- and between-day CV results, matrix effect, and process efficiency were <15%. The AAS were stable for at least 6 months at −20°C. Serum samples were obtained from previous studies. A novel finding from an administration study was that T enanthate was present in serum even after 5 years of storage at −20°C. Serum samples from self-reporting AAS individuals, where T esters were detected, were positive for testosterone using the urinary testosterone/epitestosterone criterion >10. Of those identified as positive in traditional urinary doping tests (n = 15), AAS in serum were found in 80% of the subjects. Our results show that serum may be a valid complementary matrix to urine samples for AAS testing.     

Development of an ultra‐high performance liquid chromatography–tandem mass spectrometry method for the determination of anabolic steroids currently available on the black market in the Czech Republic and Slovakia

Over the past decade, the use of anabolic androgenic steroids (AAS) by recreational bodybuilders and regular gym members has been increasing exponentially. However, there is a lack of knowledge on AAS products sold in the Czech Republic and Slovakia. The aim of this study was to assess the quantity of active substances in AAS products obtained from AAS users in the Czech Republic and Slovakia. In addition, the study also examines the current trends in counterfeit AAS products used by recreational bodybuilders and regular gym members. For this purpose, the authors developed and validated a universal ultra‐high performance liquid chromatography with tandem mass spectrometry method to determine the most popular doping substances in different pharmaceutical formulations (oil‐based injectables, tablets, and capsules). This method was successfully utilized for the analysis of 358 AAS products. Our results showed that 58.9% AAS products analyzed contained the declared active substances at the declared concentrations, 15.9% contained no active substance, 16.8% were under‐concentrated, 4.5% contained active substances different from those declared on the label, and 3.6% products were over‐concentrated. Alarmingly, the results demonstrated that over 40% of the AAS analyzed failed to meet label claims and therefore may pose serious health risks to consumers. This study also highlighted that the availability of AAS should be more rigorously restricted and their quality closely monitored in order to protect AAS users. In conclusion, the authors have developed a precise, accurate, sensitive, selective, and robust method for the routine screening of products containing anabolic androgenic steroids.     

A pilot wastewater‐based epidemiology assessment of anabolic steroid use in Queensland,Australia

Anabolic‐androgenic steroids are synthetic compounds prohibited due to their performance‐enhancing characteristics. The use of these substances is known to cause health‐related issues, which highlights the importance of being able to evaluate the scale of consumption by the general population. However, most available research on the analysis of anabolic steroids is focused on animals and athletes in connection with doping. The potential of wastewater‐based epidemiology as an intelligence tool for the assessment of community level use of anabolic steroids is presented herein. A liquid chromatography tandem mass spectrometry method was developed for the analysis of 10 anabolic‐androgenic steroids and 14 endogenous hormones in influent wastewater. The validated method was applied to sixteen 24‐hour composite wastewater influent samples that were collected over a period of five years from two wastewater treatment plants in Queensland, Australia. Nine investigated compounds were found to be present at concentrations between 14 and 611 ng L^?1 which translated into 3–104 mg excreted per 1000 individuals per day. It was concluded that the developed analytical method is suitable for the analysis of AAS in wastewater matrix. Additionally, both the inclusion of metabolites and further investigation into deconjugation by enzymatic hydrolysis would aid in understanding and evaluating community anabolic steroid use. For the first time, this study presents the application of wastewater‐based epidemiology on anabolic‐androgenic steroids in Australia.     

Bioformation of boldenone and related precursors/metabolites in equine feces and urine,with relevance to doping control

Boldenone (1‐dehydrotestosterone) is an exogenous anabolic‐androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1‐dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1‐dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1‐dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1‐progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1‐progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1‐dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1‐progesterone was also detected. Δ1‐progesterone was not detected any non‐incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1‐progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.     

Toxicological findings and manner of death in autopsied users of anabolic androgenic steroids

With the aim to characterize patterns in toxicological profile and manner of death in deceased users of anabolic androgenic steroids (AAS), a retrospective autopsy protocol study of 52 deceased users of AAS was undertaken. The AAS users were compared to 68 deceased users of amphetamine and/or heroin who were consecutively tested and found to be negative for AAS. Use of AAS was in the majority of cases (79%) associated with concomitant use of psychotropic substances. AAS-related deaths differed in several respects from deaths among users of heroin or amphetamine, most strikingly with regard to: (a) the median age at death, which was significantly lower for AAS users (24.5 years) than for users of heroin and/or amphetamine (34 and 40 years, respectively); (b) the manner of death, with AAS users dying significantly more often from homicide or suicide than users of other drugs; and (c) the body mass index (BMI), with AAS users exhibiting significantly higher BMI than users of other drugs. These results support the earlier reported association between use of AAS and use of other psychoactive substances. In addition, the data suggest that AAS users are more likely to become involved in incidents leading to violent death and have a higher risk of dying at a younger age than users of other drugs.     

Anabolic–Androgenic Steroid Use Among Brazilian Bodybuilders

This cross-sectional, quantitative, exploratory study investigated the prevalence and profile of anabolic–androgenic steroids (AAS) users amongst a convenience sample of 510 bodybuilders from 52 gyms, in João Pessoa, Brazil, with a structured questionnaire containing selected questions about socioeconomic and training variables on the use of AAS. Data were analyzed using frequency and chi-square tests. AAS prevalence use was 20.6%; mostly young men (98.1%), of a low education level (46.7%), who trained for more than 4 years (49.5%). The use of AAS was related to the use of dietary supplements. About 81% of consumed AAS consisted of Deca-Durabolin, Winstrol, and Sustanon. Study's limitations are noted.     

Abuse of anabolic androgenic steroids and related substances in sport and exercise

Anabolic androgenic steroids are synthetic derivatives of testosterone, which is the primary male sex hormone. Anabolic androgenic steroids are used to enhance athletic performance and appearance. Adverse effects include those on the liver, serum lipids, psyche/behavior and reproductive system. Androstenedione is an anabolic androgenic steroid used to increase blood testosterone levels for the purposes of increasing strength, lean body mass and sexual performance. However, there is no research indicating that androstenedione, or its related compounds, significantly increases strength and/or lean body mass in humans by increasing testosterone levels. The long-term health effects of prolonged androstenedione supplementation are unknown. Dehydroepiandrosterone (DHEA) is a weak androgen also used to elevate testosterone levels, and is advertised as an anti-obesity and anti-aging supplement capable of improving libido, vitality and immunity levels. However, research demonstrates that DHEA supplementation does not increase serum testosterone concentrations or increase strength in men, and may acutely increase testosterone levels in women, thus producing a virilizing effect.     

UDP‐glucuronosyltransferase 2B17 genotyping in Japanese athletes and evaluation of the current sports drug testing for detecting testosterone misuse

Ethnicity has been found to influence urinary testosterone glucuronide to epitestosterone glucuronide (T/E) ratios among athletes. Uridine diphospho‐glucuronosyltransferase 2B17 (UGT2B17) is the most active enzyme in testosterone glucuronidation. UGT2B17 polymorphism analysis is rarely performed in Japanese athletes, and the influence of testosterone administration on steroid profiles and carbon isotope ratios, according to gene polymorphisms, in Asians remains unknown. The prevalence of UGT2B17 genotypes and urinary androgenic steroid profiles, classified according to UGT2B17 genotypes, was investigated in Japanese athletes (255 male and 256 female). Testosterone enanthate (100 mg) was administered intramuscularly to Japanese female volunteers (del/del: n = 6, del/ins: n = 3, ins/ins: n = 1). The distribution rates of the UGT2B17 del/del genotype in Japanese male and female athletes were 74.5% and 60.2%, respectively. The ins/ins genotype was detected in only three male (1.2%) and seven female (2.7%) athletes. The prevalence of the UGT2B17 deletion genotype was extremely high in Japanese athletes. The T/E ratio in the del/del group was significantly lower than that in the other groups. After testosterone was administered to female volunteers, the T/E ratios for the del/del individuals failed to reach the positivity criterion of 4. By contrast, in all of the del/del subjects, the gas chromatography/combustion/isotope ratio mass spectrometry (GC‐C‐IRMS) analysis successfully fulfilled the positivity criterion. The overall result has demonstrated the limited effectiveness of population‐based T/E ratios in screening tests for testosterone use. Subject‐based steroid profiling with UGT2B17 genotyping will be an effective strategy for detecting testosterone misuse. Copyright © 2012 John Wiley & Sons, Ltd.     

Vulnerability and the Intention to Anabolic Steroids Use Among Iranian Gym Users: An Application of the Theory of Planned Behavior

This correlational study explored the psychological antecedents of 253 Iranian gym users’ intentions to use the anabolic–androgenic steroids (AAS), based on the Theory of Planned Behavior (TPB). The three predictor variables of (1) attitude, (2) subjective norms, and (3) perceived behavioral control accounted for 63% of the variation in the outcome measure of the intention to use the AAS. There is some support to use the TPB to design and implement interventions to modify and/or improve individuals’ beliefs that athletic goals are achievable without the use of the AAS.     

Evaluation of longitudinal steroid profiling with the ADAMS adaptive model for detection of transdermal,intramuscular, and subcutaneous testosterone administration

The steroidal module of the Athlete Biological Passport (ABP) has been used since 2014 for the longitudinal monitoring of urinary testosterone and its metabolites in order to identify samples suspicious for the use of synthetic forms of endogenous anabolic androgenic steroids (EAAS). Samples identified by the module may then be confirmed by isotope ratio mass spectrometry (IRMS) to establish clearly the exogenous origin of testosterone and/or metabolites in the sample. To examine the detection capability of the steroidal ABP model, testosterone administration studies were performed with various doses and three routes of administration – transdermal, intramuscular, and subcutaneous with 15 subjects for each route of administration. Urine samples were collected before, during, and after administration and steroid profiles were analyzed using the steroidal ABP module in ADAMS. A subset of samples from each mode of administration was also analyzed by IRMS. The steroidal ABP module was more sensitive to testosterone use than population‐based thresholds and with high dose administrations there was very good agreement between the IRMS results and samples flagged by the module. However, with low dose administration the ABP module was unable to identify samples where testosterone use was still detectable by IRMS analysis. The testosterone/epitestosterone (T/E) ratio was the most diagnostic parameter for longitudinal monitoring with the exception of low testosterone excretors for whom the 5α‐androstane‐3α, 17β‐diol/epitestosterone (5αAdiol/E) ratio may provide more sensitivity.     

Abnormal cardiac repolarization in anabolic androgenic steroid users carrying out submaximal exercise testing

1. The aim of the present study was to investigate the cardiovascular effects of anabolic androgenic steroid (AAS) abuse by comparing the electrocardiographic parameters before and after submaximal exercise between AAS users and non-AAS users. 2. A total of 22 men who regularly engaged in both resistance and aerobic exercise at fitness academies volunteered for the study (control group: n = 11, age 25 ± 4 years; AAS group: n = 11, age 27 ± 5 years). All subjects were submitted to submaximal exercise testing using an Astrand-Rhyming protocol. Heart rate and electrocardiography parameters were measured at rest and at the third minute of the post-exercise recovery time. 3. AAS users presented higher QTc and QTd at rest (10% and 55%, respectively) and at the post-exercise period (17% and 43%, respectively), compared with control subjects. The maximal and minimum QTc interval of the AAS group was significantly prolonged at the post-exercise period (12% and 15%, respectively). The haemodynamic parameters were similar in both groups (P > 0.05). The AAS group showed a lower heart rate recovery at the first minute after the test (P = 0.0001), and a higher exertion score (P < 0.0001) at a lower workload, compared with the control group. 4. Our results show that the QTc interval and dispersion are increased in individuals who abuse AAS, suggesting the presence of ventricular repolarization abnormalities that could potentially increase the risk of cardiac arrhythmias and sudden cardiac death.     

Behavioural manifestations of anabolic steroid use

The use of anabolic androgenic steroids (AAS) for gains in strength and muscle mass is relatively common among certain subpopulations, including athletes, bodybuilders, adolescents and young adults.Adverse physical effects associated with steroid abuse are well documented, but more recently, increased attention has been given to the adverse psychiatric effects of these compounds. Steroids may be used in oral, 17alpha-alkylated, or intramuscular, 17beta-esterified, preparations. Commonly, steroid users employ these agents at levels 10- to 100-fold in excess of therapeutic doses and use multiple steroids simultaneously, a practice known as 'stacking'. Significant psychiatric symptoms including aggression and violence, mania, and less frequently psychosis and suicide have been associated with steroid abuse. Long-term steroid abusers may develop symptoms of dependence and withdrawal on discontinuation of AAS. Treatment of AAS abusers should address both acute physical and behavioural symptoms as well as long-term abstinence and recovery. To date, limited information is available regarding specific pharmacological treatments for individuals recovering from steroid abuse. This paper reviews the published literature concerning the recognition and treatment of behavioural manifestations of AAS abuse.     

Use of anabolic androgenic steroids in substance abusers arrested for crime

Background

Use of anabolic androgenic steroids (AASs) has been associated with both violent crime and the use of illicit drugs. The scientific literature on polysubstance abuse as a confounder for AAS-related violence is sparse and ambiguous. With the intent of further investigating this issue, we have gathered data concerning drug abuse and AAS experience among substance abusers who have been arrested for a variety of crimes.

Methods

Data were collected from structured interviews with substance abusers (n = 3597) apprehended at two remand prisons in Sweden from 2002 through 2008. Analyses concerned type of criminal act, primary drug used during the past year, and experience of AAS use.

Results

Those stating AAS experience (n = 924, 20 women and 904 men) were more often apprehended for violent crimes (OR = 1.65). This association remained significant after controlling for age and sex (OR = 1.28). AAS users and non-users claimed similar primary substances of use during the past year, with the exception of benzodiazepine use, which was more common in the AAS group (OR = 2.30), although this did not affect the frequency of violent crime. Among AAS-experienced participants, there was no difference in violent crime incidence between current users and former users.

Conclusions

Study results suggest that AASs do not function as a proximal trigger for violence but still involve an increased risk for violence in users of illicit drugs. These findings also suggest that AAS use is highly overrepresented in women who commit crimes.     

Unambiguous identification and characterization of a long‐term human metabolite of dehydrochloromethyltestosterone

In doping control analysis, the characterization of urinary steroid metabolites is of high interest for a targeted and long‐term detection of prohibited anabolic androgenic steroids (AAS). In this work, the structure of a long‐term metabolite of dehydrochloromethyltestosterone (DHCMT) was elucidated. Altogether, 8 possible metabolites with a 17α‐methyl‐17β‐hydroxymethyl – structures were synthesized and compared to a major DHCMT long‐term metabolite detected in reference urine excretion samples. The confirmed structure of the metabolite was 4α‐chloro‐18‐nor‐17β‐hydroxymethyl‐17α‐methyl‐5α‐androst‐13‐en‐3α‐ol.     

Evaluation of longitudinal steroid profiles from male football players in UEFA competitions between 2008 and 2013

Testosterone and related compounds are the most recurrent doping substances. The steroid profile, consisting of the quantification of testosterone and its metabolites, has been described as the most significant biomarker to detect doping with pseudo‐endogenous anabolic steroids. The steroidal module of the Athlete Biological Passport (ABP) was launched by the World Anti‐Doping Agency (WADA) in 2014. To assess the value of introducing the module to its anti‐doping programme, the Union of European Football Associations (UEFA) decided to analyze retrospectively the steroid profile data of 4195 urine samples, collected from 879 male football players and analyzed in 12 WADA‐accredited laboratories between 2008 and mid‐2013. This study focused on the evaluation of T/E ratios. The coefficient of variation (CV) and the adaptive model were the two statistical models used to study the longitudinal follow‐up. A CV of 46% was determined to be the maximal natural intra‐individual variation of the T/E when the sequence consisted of single data points analyzed in different laboratories. The adaptive model showed some profiles with an atypical T/E sequence and also enabled an estimate of the prevalence of external factors impacting the T/E sequences. Despite the limitations of this retrospective study, it clearly showed that the longitudinal and individual follow‐up of the T/E biomarker of the players is a good tool for target testing in football. UEFA has therefore decided to implement the steroidal module of the ABP from the start of the next European football season in September 2015. Copyright © 2015 John Wiley & Sons, Ltd.     

Outline of a typology of men’s use of anabolic androgenic steroids in fitness and strength training environments*

Recent research into the use of anabolic androgenic steroids (AAS) in fitness and strength training environments have revealed great variance in users’ approach to AAS use and more specifically their approach to health risks and desired objectives. However, there have only been few attempts to develop theoretical frameworks directed at conceptualising the variance in AAS use. In this paper, we propose a unified framework in the form of a typology, which concerns men’s general approach to AAS use. The typology is based on sociologist Max Weber’s method on the ideal typology. The work comes out of the authors’ own qualitative empirical research on male AAS users in fitness and strength training environments, but is also related to and draws on the international literature on the subject. The suggested typology consists of four ideal types: the Expert type, the Well-being type, the YOLO type and the Athlete type. The four types are developed around two overarching categories, namely users’ approach to risk and effectiveness. The typology outlines distinct and characteristic approaches to AAS use and can, thus, be employed by researchers as well as health professionals as a heuristic tool for investigation and explanation.     

Long-term stability study and evaluation of intact steroid conjugate ratios after the administration of endogenous steroids

The most frequently detected substances prohibited by the World Anti-Doping Agency (WADA) belong to the anabolic steroids class. The most challenging compounds among this class are the endogenous anabolic steroids, which are detected by quantitative measurement of testosterone (T) and its metabolites with a so-called “steroid profiling” method. The current steroid profile is based on the concentrations and ratios of the sum of free and glucuronidated steroids. Recently, our group developed a steroid profiling method for the detection of three free steroids and 14 intact steroid conjugates, including both the glucuronic acid conjugated and sulfated fraction. The study aimed at evaluating the long-term stability of steroid conjugate concentrations and ratios, and the influence of different endogenous steroids on this extended steroid profile. A single dose of oral T undecanoate (TU), topical T gel, topical dihydrotestosterone (DHT) gel, and oral dehydroepiandrosterone (DHEA) was administered to six healthy male volunteers. One additional volunteer with a homozygote deletion of the UGT2B17 gene (del/del genotype) received a single topical dose of T gel. An intramuscular dose of TU was administered to another volunteer. To avoid fluctuation of steroid concentrations caused by variations in urinary flow rates, steroid ratios were calculated and evaluated as possible biomarkers for the detection of endogenous steroid abuse with low doses. Overall, sulfates do not have substantial additional value in prolonging detection times for the investigated endogenous steroids and administration doses. The already monitored glucuronides were overall the best markers and were sufficient to detect the administered steroids.     

Highly sensitive avidin‐biotin ELISA for detection of nandrolone and testosterone in dietary supplements

Avidin‐biotin technology was used for the implementation of an enzyme‐linked immunosorbent assay (AB‐ELISA) as a sensitive method for the detection of anabolic androgenic steroids (AAS) present in dietary supplements. Using click chemistry, novel haptens (linker‐optimized biotinylated nandrolone (NT) and testosterone (T) at positions C‐3 and C‐17, respectively) were designed and synthesized to be then applied as four different immobilized competitors in a proposed set of four indirect competitive AB‐ELISAs. Four rabbit polyclonal antibodies of various specificities were prepared using four different immunogens synthesized from C‐3 and C‐17 carboxymethyloxime and hemisuccinate derivatives of NT and T, respectively. Assembled AB‐ELISAs were characterized to establish method parameters such as a half‐maximum inhibition concentration (0.18–12.99 ng/mL), limit of detection (0.004–0.032 ng/mL) and linear working range (the best with 0.02–1.38 ng/mL). The stability of the set simulating storage in different conditions was demonstrated. Cross reactivity (CR) was tested for 59 steroids including both endogenous and synthetic analogues in four assembled AB‐systems. The focus was placed on the practical use of the method in detection of various AAS in 49 samples of counterfeit dietary supplements. The concordance between ultra high performance liquid chromatography‐mass spectrometry (UHPLC‐MS) and the CR corrected data from AB‐ELISA indicated the potential of this method even to quantification of T propionate, NT phenyl propionate, and NT decanoate in such a complex matter. Copyright © 2016 John Wiley & Sons, Ltd.